Research Journal of Biotechnology

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Research Journal of Biotechnology

Genetic and phenotypic diversity among local okra (Abelmoschus esculentus L.) landraces using AFLP markers

Akash Muhanad, Abu Hussein Mahmoud M., Al-Abdallat Ayed M., Shiyab Safwan and Zatimeh Ahmad

Page No: 1-13

Abstract: Genetic and phenotypic variability among 21 okra (Abelmoschus esculentus L.) landraces were explored. Thirteen horticultural traits were studied including fruit, stem and petiole color, spine existence, fruit length, fruit diameter, number of ridges per fruit, seeds number per fruit, days to 50% flowering, plant height, shoot fresh weight, shoot dry weight and number of fruits per plant. Except for fruit diameter, plant height and number of fruits per plant, a significant variability among landraces was found. Genetic variability and relationships among the 21 landraces were established using twelve AFLP primer combinations. Six hundred bands with 162 polymorphic bands were sufficient to estimate genetic variation among okra landraces.

The unweighted pair group method with arithmetic average classified landraces into three groups was based on Dice similarity coefficient, which ranged from a high of 0.99 to a low of 0.50. Clustering based on phenotypical traits showed seven smaller groups that were consistent with the three larger AFLP-based groups. These diverse landraces can be used for future okra breeding programs and derivation of new lines.

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Molecular characterization and cross infectivity of poty and begomo viruses associated with hot pepper (Capsicum annuum L.) in Punjab (India)

Kaur Sukhjeet, Kang Santokh Singh and Sharma Abhishek

Page No: 14-22

Abstract: Virus diseases of hot pepper (Capsicum annuum L.) are one of the major limiting factors in hot pepper production causing serious economic losses. Under Punjab conditions, hot pepper remains in field up to nine months. During this long cropping period, different types of virus symptoms were exhibited by the crop. Among these symptoms vein banding, mosaic, puckering and curling of leaves were most predominating.

Incidence of Pepper mottle potyvirus was recorded first time in India from Ludhiana, Punjab, however information on its distribution in major pepper growing areas of Punjab was lacking. Distribution and occurrence of Pepper mottle virus (PepMoV) along with begomoviruses in different pepper growing districts of Punjab were conducted to systematically analyse the samples collected from the major pepper growing districts of Punjab using Double Antibody Sandwich-Enzyme Linked Immunosorbant Assay (DAS-ELISA), RT-PCR and Imunocapture RT-PCR for RNA viruses and Polymerase Chain Reaction (PCR) with coat protein specific primers for begomoviruses.

Presence of PepMoV was recorded from all the five major hot pepper growing districts (Ludhiana, Patiala, Tran Taran, Sangrur and Ferozepur) of Punjab. Among the begomoviruses, prevalence of Tomato leaf curl Joydebpur virus and Pepper leaf curl virus was observed. Tomato leaf curl Joydebpur virus has been found prevalent in Tarn Taran, Ferozepur, Sangrur and Patiala districts and Pepper leaf curl virus in Ludhiana district. The phylogenetic analysis revealed that the PepMoV isolate infecting pepper in Punjab shared maximum similarity with PepMoV isolates from California and Florida. Cross infectivity studies done on PepMoV and Tomato leaf curl Joydebpur virus from hot pepper to other solanaceous vegetable crops showed that both the viruses can easily be transmitted to tomato, brinjal, potato and sweet pepper.

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Hypolipidemic effect of Sargassum swartzii extract in macrophage and hepatocyte cells mediated by the regulation of peroxisome proliferator activated receptor α and γ

Hoang Thi Minh Hien, Nguyen Cam Ha, Ngo Thi Hoai Thu and Dang Diem Hong

Page No: 23-27

Abstract: In this study, we demonstrated that Sargassum swartzii extract (SE) is a dual agonist for PPARα and γ, identified from reporter gene assay and qPCR analysis. Cultured macrophages and hepatocytes stimulated with SE at 100 µg/mL exhibited significantly reduced cellular cholesterol and triglyceride concentrations while cellular uptake of fatty acids in hepatocytes was increased by 217% (P < 0.05). Quantitative PCR analysis revealed that the increase in cellular fatty acid uptake was due to up-regulation of fatty acid transporter protein-4 (+ 101% vs. the control) in hepatocytes stimulated with SE at 100 g/mL.

Moreover, expression of genes related to fatty acid oxidation and ketogenesis and cholesterol efflux were up-regulated, while that of genes related to fatty acid synthesis were suppressed in cells treated with SE. These results suggest that SE is a direct ligand for PPARα and γ with the ability to decrease lipid concentrations by regulating the expression of lipid metabolism genes.

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Molecular characterization and tissue expression of common tobacco (Nicotiana tabacum) cadmium resistance protein 10 and 12 genes

Liu Yonggang and Kong Chuisi

Page No: 28-33

Abstract: The full-length coding regions of common tobacco cadmium resistance protein 10 and 12 genes were cloned by RT-PCR. The tobacco cadmium resistance protein 10 gene encode a protein of 471 amino acids which shares high identity with thecadmium resistance protein 10 of five species—nicotiana tomentosiformis (100%), wood tobacco (98%), lycopersicon pennellii (93%), lycopersicon esculentum (93%) and potato (89%). The tobacco cadmium resistance protein 12 gene encodes a protein of 399 amino acids which shares high identity with thecadmium resistance protein 12 of four species—nicotiana tomentosiformis(99%), wood tobacco (97%), lycopersicon esculentum (83%) and potato (81%). Prediction of transmembrane helices showed that cadmium resistance protein 10 and 12 might be two transmembrane proteins. Phylogenetic analysis revealed that the common tobacco ocadmium resistance protein 10 and 12 genes both have a closer genetic relationship with the nicotiana tomentosiformis cadmium resistance protein 10 and 12 genes.

Computer-assisted analysis showed that cadmium resistance protein 10 gene is structured in 3 exons and 2 introns. Cadmium resistance protein 12 gene is structured in 4 exons and 3 introns. Tissue expression profile analysis indicated that the common tobacco cadmium resistance protein 10 and 12 genes were highly expressed in root.

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Gene action conditioning resistance to Aspergillus ear rot in maize under water stress conditions

Manal Hefny, Metwali E., Ali A., El-Marzouki H. and Isam Abu Zeid

Page No: 34-43

Abstract: The exploitation of genetic variability to develop new sources resistant to Aspergillus ear rot is a prime concern for corn breeders. The present study was designed to estimate gene action and inheritance for flowering traits, ear rot percent and antioxidants activities of five maize inbred lines and their 10 diallel single crosses under control and water stress conditions. Two irrigation regimes; control and water stress and the 15 genotypes were allocated to main and sub-plots respectively. General (GCA) and specific (SCA) combining abilities were significant sources of variation, although GCA recorded greater contribution of the variation. The crosses INB45xINB39, INB76xINB39, INB31xINB76 and INB31xINB39 with negative and significant SCA effects for low ear rot infection were considered useful in future breeding programs targeting both reduced levels of fungal infection and early silking. Using ISSR marker, a total of eight bands were found to be useful positive or negative markers of drought stress.

The cluster analysis based on ISSR analysis clearly distinguished maize genotypes from each other while cluster A, B and C included tolerance parental genotypes, while sensitive genotypes were included in cluster E with most of their hybrids, that reflect its different genetic structure from other tested maize genotypes.

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Effect of Ultrasonic on salting and quality changing of Marinated Eggs

Wu Hui-qing, Wei Shuai, Jin Yong-guo and Ma Mei-hu

Page No: 44-51

Abstract: In this paper, egg white protein gels of marinated eggs were treated by high frequency ultrasound (40Khz, 150W) and the effects on moisture changes, salt diffusion and texture quality were studied. Low-field NMR results indicated that T22 (non-flowing water) was the major form (98%) of water in marinated egg gels. When the ultrasonic time was increased, the combination of the gel and water became weak. Further, the total proton density of egg white protein gels was decreased and the moisture content was significantly reduced.

SEM results showed that ultrasound increased the pore size of the gel so that infiltration and diffusion of salt were promoted which improved the salting efficiency by 91.67%. After ultrasonic treatment, new pore structures came out that made the gel structure more compact. The gel hardness, chewiness and adhesion were significantly improved after 2-8 h treatment by ultrasonic. In summary, after ultrasonic treatment, the water content of marinated eggs decreased, thus, the storage property was improved; the infiltration of salt was promoted so that the salting efficiency became higher; the hardness, chewiness and adhesiveness of the gel were improved so the texture quality of marinated egg was promoted.

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Elucidation of genetic diversity among ashwagandha [Withania somnifera (L.) Dunal] genotypes using EST-SSR markers

Barvaliya Parita, Singh Nirbhay Kumar, Dharajiya Darshan and Pachchigar Karen

Page No: 52-59

Abstract:Ashwagandha [Withania somnifera (L.) Dunal] is one of the most commonly used herbs in Indian traditional medicine. The genomic DNA of 36 ashwagandha genotypes was extracted by modified CTAB method to evaluate diversity through EST-SSR markers. Total nine polymorphic primers were used for the screening across all the genotypes of ashwagandha which amplified 55 polymorphic bands. Polymorphic Information Content (PIC) of each marker varied from 0.21 to 0.86. The mean PIC, per cent polymorphism and resolving power were 0.732, 94.71 and 3.23 respectively. The Jaccard´s dissimilarity co-efficients were able to classify all the genotypes of ashwagandha into three clusters where cophenetic correlation coefficient was 0.933.

Genetic similarity among the genotypes ranged from 0.069 to 0.846 indicating high genetic divergence among the genotypes used in the present study. Ashwagandha genotypes MWS-205-3-2 and MWS-322-1-2 had the highest similarity while genotypes MPAS-15-3-1 and RAS-23-2-1 had minimum similarity. Further, from NJ analysis, RAS-11-3-2 could be the most primitive genotype among all the genotypes used in the study. Hence, EST-SSRs derived from solanaceae family can be used for genetic diversity analysis in Withania somnifera due to their cross-species transferability.

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Sensitivity and Stability Analysis of Reproduction Number on the Epidemic Model of Ebola Virus in comparing Environmental Changes with the Critical Immunity Levels in Population Size

Radha M. and Balamuralitharan S.

Page No: 60-64

Abstract: The Ebola Hemorrhagic Fever (EHF) that causes a highly virulent infectious disease, has plagued West Africa, Liberia, Leone and Guinea heavily in 2014. We use an epidemic model to simulate the transmission of the disease which has immunized susceptible(S), exposed (E), infectious (I) and a recovered population(R). Systematically a disease-free equilibrium state exists all the time for epidemic model. Instability of the disease-free equilibrium state implies the endemic equilibrium state.

We analyzed the model parameters for the sensitivity index for changes in the disease-free reproduction number Rdf. Critical immunization level obtained from the basic reproduction number R0which is important to avoid the spread of virus in disease-free state. From the sensitivity analyses, the most important parameters are recognized to control the spread of virus in an infected population.

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Larvicidal Activity of Synthesized Silver Nanoparticles using Rhazya stricta Leaf Extract against Mosquito Vectors Aedes Aegypti

Alyaha Hanan S., Mahyoub Jazem A., Ghramh Hamed A. and Alhag Sadeq K.

Page No: 65-72

Abstract: The synthesis of silver nanoparticles (AgNPs) using plant extracts is fulfilled and considered as ecofriendly method. The Aedes aegypti mosquito is a vector of several diseases in humans like yellow fever and dengue. There is no specific treatment for dengue. Its prevention and control solely depend on effective vector control measures. The purpose of this study was to investigate the larvicidal activity of the ethanol leaf extracts of Rhazya stricta and synthesized silver nanoparticles against 4th instar larvae of Ae. aegypti. The experimented excuse was according to the WHO protocols. R. stricta and Synthesized silver nanoparticles (AgNPs).

Particles were characterized by analyzing the excitation of surface plasmon resonance (SPR) using UV–vis spectrophotometer and FT-IR spectra. Further TEM analysis confirmed the range of particle of nanoparticles. Our results showed a high efficiency of the R. stricta extract where the fatal concentration is for 50 % of the population (LC50) (323,447 ppm). Moreover, with addition of the silver nano partials the (LC50) was 91,342 ppm. The nanoparticles were more lethal against 4th instar larvae of Ae. aegypti by about 3.46 folds. These results suggest that the synthesized AgNPs of R. stricta have the potential to be used as an ideal eco-friendly compound for the control of the Ae. aegypti larvae.

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Screening and isolation of therapeutic agent for type 2 diabetes mellitus from endophytic fungal metabolites of mangrove plants

Nathiya R. and Gayathri Mahalingam

Page No: 73-86

Abstract: To investigate the antidiabetic activity of metabolite from novel strain of endophytic fungi isolated from four different mangrove plants namely Avicennia marina, Acanthus ilicifolius, Rhizophora mucronata and Rhizophora apiculata. The crude extracts of three different solvents were extracted and analyzed for α-glucosidase and α-amylase inhibitory assay done with various concentrations (250, 500, 750 and 1000 µg/ml) and were compared with standard drug acarbose. The MTT assay was done on L929 cell lines of the crude extract to predict its cytotoxic effect. The results showed that crude extract of VITNG3 strain had prominent α-glucosidase and α-amylase activity with an inhibition percentage of 73.7±1.25 and 32.17±1.18 in an IC50 range of 367.6 µg/ml and 101.0 µg/ml respectively.

Further, the cytotoxic effect on L929 cell lines of the EtOAc crude extract of Collectotrichum siamense starin VITNG3 showed a viability of 74.33% at 1000 μg/ml concentration and thus it was considered as less toxic. A new strain, Collectotrichum siamense starin VITNG3 was isolated from the leaf of Acanthus ilicifolius had satisfactory anti-diabetic activity for type 2 diabetes, which will further be confirmed by in vivo studies.

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Inhibition of Calcium Oxalate Crystallization in vitro by an extract of Kigelia africana

Sharma Mohit, Bala Anoop Sirish Kataru, Vigneshwari T., Sravani N., Vinodhini S. and Devi Rajeswari V.

Page No: 87-91

Abstract: To investigate the effectiveness of Kigelia africana methanolic extract on the inhibition of crystallization in synthetic urine, calcium oxalate (COM) and sodium oxalate solutions were induced together to form crystallization as synthetic urine. Methanolic extract of Kigelia africana was prepared in four different concentrations (25, 50, 75, 100µg/ml). An analysis was done to verify the turbidity changes during the crystal nucleation and the aggregation in the solution of synthetic urine was observed at 620nm by spectrophotometer and standard was used as a cystone. Methanolic extract was tested against the artificial stone of calcium oxalate crystals by homogeneous precipitation method.

This study was assessed for a crystal dissolution using the semi-permeable membrane of the egg which is used as dissolution bag and the estimation was done using a spectrophotometric method. The result shows that extract of Kigelia africana has greater capability to dissolve the calcium oxalate compared to the standard of cystone that shows better activity in dissolution model. Kigelia africana extract and cystone exhibited strong inhibitory activity in the nucleation and aggregation assays. We concluded that Kigelia africana leaf extract remarkably inhibits the crystal formation and it has good potential for the anti-urolithiatic activity. Hence, further investigation and formation of new drugs from this medicinal plant can be used for urolithiasis.

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Elicitor Mediated enhanced Caulogenesis and reduced contamination during Micropropagation in Ocimum sanctum L.

Sharma Vikas, Kumari Kavita, Chaudhary Priyanka and Sharma Shivika

Page No: 92-97

Abstract: Ocimum sanctum L. commonly known as “Holy Basil” belongs to family Lamiaceae. It is not only valued for its pharmaceutical properties but also for its aromatic oils and has enormous potential in cosmetic and drug industry. In present study an alternative protocol for in vitro propagation of Ocimum sanctum was developed using PGRs, additives and biotic elicitor and their effect on in vitro caulogenesis was studied. Shoot multiplication was initiated from nodal explants cultured on MS medium fortified with different concentrations of Indoleacetic acid (0.1 to 2.0 mg/l), α-Naphthalene-acetic acid (0.1 to 2.0 mg/l), 6-Benzyl-aminopurine (0.1 to 2.0 mg/l), Biotic elicitor (Yeast Elicitor) (0.25 to 2.0 mg/l), Additives (Adenine sulphate) (0.001 to 0.005%).

It has been observed that PGRs, additives and elicitors at different concentration brought out significant morphogenic variation in caulogenesis. The maximum number of shoots (16 ±0.57) and (19.1 ± 0.13) was achieved with 1mg/l BAP alone and in combination with 0.5mg/l IAA respectively. Maximum number of shoots (20.16± 0.38) and (24.0 ± 0.57) was achieved with 0.005% adenine sulphate and 0.75 mg/l yeast elicitor respectively. It was notified that yeast elicitor reduced the chances of microbial attack as compared to other treatments.

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In silico identification of mi-RNAs from Mango (Mangifera indica L.) fruit transcriptome data

Ahmad Israr, Muthukumar Manoharan, Hudedamani Umesh and Rajan Shailendra

Page No: 98-104

Abstract: Mango (Mangifera indica L.) occupies vital place among the fruit crops grown in India and is considered as the king of fruits, owing to its delicious flavour and taste. Till date no information regarding small RNA and that of the genomic regions governing them has been characterized in mango. It is evident that miRNAs plays crucial role in gene expression and regulation of complex biological mechanisms in different plant species. In the present study, through in silico analysis a total of 18 conserved miRNAs belonging to 15 families were identified from the small RNAs shortlisted from the transcriptome data set available from NCBI database. Some of the important miRNAs identified in the study include min-miRNA 159, min-miRNA 319, min-miRNA 397, min-miRNA160 and min-miRNA393.

The potential targets predicted were related to genes that are involved in a wide range of metabolic and regulatory pathways. GO (Gene Ontology) enrichment analysis revealed that cellular and metabolic pathways are the most significant pathways with respect to the rich factor and gene numbers. These miRNAs identified in this study were found to be either involved in development, cell wall metabolism, transport and/or signalling pathways.

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Isolation and identification of BHV-1 from nasal swab specimens of cattle: A trial study

Ganguly Subha and Das Ratna

Page No: 105-106

Abstract: The present study was undertaken to isolate and identify bovine herpes virus-1 (BHV-1) from nasal swabs collected randomly from cattle. No positive BHV-1 was able to be isolated and identified in the present trial.

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